Reading multi-featured arrays

ABSTRACT

A method, apparatus, and computer program product for reading fluorescence signals from an array of chemical moieties (such as different sequence peptides or polynucleotides, for example different DNA sequences). In the method the spatial sequence of scanned locations need not be the same as the temporal sequence. For example, a later illuminated line may be spatially closer to an earlier illuminated line than is a temporally intervening illuminated line.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of application Ser. No.09/846,125 filed on Apr. 30, 2001 and now issued as U.S. Pat. No.6,756,202; the disclosure of which is herein incorporated by reference.

FIELD OF THE INVENTION

This invention relates to arrays, particularly biopolymer arrays such asDNA or protein arrays, which are useful in diagnostic, screening, geneexpression analysis, and other applications.

BACKGROUND OF THE INVENTION

Polynucleotide arrays (such as DNA or RNA arrays) and peptide array, areknown and may be used, for example, as diagnostic or screening tools.Such arrays include regions (sometimes referenced as spots or features)of usually different sequence polynucleotides or peptides arranged in apredetermined configuration on a substrate. The array is “addressable”in that different features have different predetermined locations(“addresses”) on a substrate carrying the array.

Biopolymer arrays can be fabricated using in situ synthesis methods ordeposition of the previously obtained biopolymers. The in situfabrication methods include those described in U.S. Pat. No. 5,449,754for synthesizing peptide arrays, and in U.S. Pat. No. 6,180,351 and WO98/41531 and the references cited therein for polynucleotides. In situmethods also include photolithographic techniques such as described, forexample, in WO 91/07087, WO 92/10587, WO 92/10588, and U.S. Pat. No.5,143,854. The deposition methods basically involve depositingbiopolymers at predetermined locations on a substrate which are suitablyactivated such that the biopolymers can link thereto. Biopolymers ofdifferent sequence may be deposited at different feature locations onthe substrate to yield the completed array. Procedures known in the artfor deposition of polynucleotides, particularly DNA such as wholeoligomers or cDNA, are described, for example, in U.S. Pat. No.5,807,522 (touching drop dispensers to a substrate), and in PCTpublications WO 95/25116 and WO 98/41531, and elsewhere (use of an inkjet type head to fire drops onto the substrate).

In array fabrication, the quantities of DNA available for the array areusually very small and expensive. Sample quantities available fortesting are usually also very small and it is therefore desirable tosimultaneously test the same sample against a large number of differentprobes on an array. These conditions require the manufacture and use ofarrays with large numbers of very small, closely spaced features.

The arrays, when exposed to a sample, will exhibit a binding pattern.The array can be read by observing this binding pattern by, for example,labeling all targets such as polynucleotide targets (for example, DNA),in the sample with a suitable label (such as a fluorescent compound),scanning an illuminating beam across the array and accurately observingthe fluorescent signal from the different features of the array.Assuming that the different sequence polynucleotides were correctlydeposited in accordance with the predetermined configuration, then theobserved binding pattern will be indicative of the presence and/orconcentration of one or more polynucleotide components of the sample.Peptide or arrays of other chemical moieties can be used in a similarmanner. Techniques and apparatus for scanning chemical arrays aredescribed, for example, in U.S. Pat. No. 5,763,870 and U.S. Pat. No.5,945,679. Apparatus which reads an array by scanning an illuminatingbeam by the foregoing technique are often referred to as scanners andthe technique itself often referred to as scanning.

Array scanners typically use a laser beam as a light source, which isscanned over the array features. A detector (typically a fluorescencedetector) with a very high light sensitivity is normally desirable toachieve maximum signal-to-noise in detecting hybridized molecules,particularly in array scanners used for DNA sequencing or geneexpression studies. At present, photomultiplier tubes (“PMTs”) are stillthe detector of choice although charge coupled devices (“CCDs”) can alsobe used. PMTs are typically used for temporally sequential scanning ofarray features, while CCDs permit scanning many features in parallel(for example, one line of features simultaneously, in which case anilluminating line may be used).

During scanning of an array, triplet saturation occurs. That is,fluorescent species are normally excited to a state from which theyreturn to the singlet ground state while emitting the fluorescent light.However, the excited species has a finite probability of ending up in alowest triplet state. Species in the triplet state do not emitfluorescence and thus are lost to producing a fluorescent signal whileremaining in that state. Unfortunately, such triplet states may havevery long lifetimes. Saturation is discussed in more detail, forexample, in U.S. Pat. No. 5,945,679. As any region containing afluorescent species may undergo multiple excitations during scanning, anincreasing proportion will be unavailable to produce a signal due tosaturation. A known solution to this problem is to re-scan a line on thearray after waiting for a sufficient time for the fluorescent species torecover from saturation. However, the present invention realizes thatfor an array containing thousands of features, this may lengthen thescanning process. Furthermore, the present invention realizes that otherproblems may arise depending upon the scanning pattern. For example, ina rectangular scanning pattern a line is scanned from a first end to asecond, and the next line is scanned from the second end to the first,and the process repeated for subsequent lines in turn. In this pattern,the time that a location has to recover from saturation is shortertoward the beginning and end of a scan line than at the center. Thepresent invention realizes that this may lead to incomplete recoveryfrom saturation and thus to a non-uniformity in detected signals from auniform array.

It would be desirable then, to provide a means to scan an array in whichthe effect of saturation can be at least reduced.

SUMMARY OF THE INVENTION

The present invention then, provides a method of reading fluorescencesignals from an array of chemical moieties (such as different sequencepeptides or polynucleotides, for example different DNA sequences). Inone such method multiple locations on the array are illuminated and anyresulting fluorescence from the array is detected. In this case a laterilluminated location is spatially closer to an earlier illuminatedlocation than is a temporally intervening illuminated location lying ona same line as the later and earlier illuminated locations. Thisprocedure may be repeated in one or more further cycles as required andusing other locations, until the array or a desired portion of it hasbeen read. Alternatively, multiple paths across the array may beilluminated and any resulting fluorescence from the array is detected.In such case the paths extend in a same lengthwise direction and arespaced from one another in a crosswise direction, and the spatialsequence of the paths does not correspond to their temporal sequence.For example, at least one later illuminated path may be closer to anearlier illuminated path than a temporally intervening illuminated path.The multiple paths may be parallel lines.

In the method, one or more later illuminated locations or paths may beinterleaved between one or more previously illuminated locations orpaths. The time between illuminating a location or path and illuminatinga closest later illuminated location or path, may be selected based on asaturation characteristic of a fluorophore producing the fluorescence.Alternatively, the time may be based on an identifier associated withthe array (such as being carried on an array substrate or a housing forthe array), or on a spatial distribution of the illumination and a pixelsize during the detecting.

Spacings of illuminated locations can be selected between any of thelocations as desired. For example, the spacing between the spatiallynearest locations or paths of the earlier, temporally intervening, andinterleaved locations or paths may be equal. Further, variousillumination techniques can be used within the methods of the presentinvention. For example, when the paths are lines they may be illuminatedby scanning a light beam along them. As another example, timewisesuccessively illuminated lines during any one cycle may be illuminatedby scanning a light beam in the same or in opposite directions. Thepresent invention further provides an apparatus which can execute amethod of the present invention. The apparatus includes an illuminationsource to cause fluorescence of the chemical moieties. A scan systemdirects the illumination source to different locations on the array,while a detector detects any resulting fluorescence from the array. Aprocessor controls the scan system to obtain the illumination of amethod of the present invention. A computer program product for use withan apparatus, for reading fluorescence signals from an array of chemicalmoieties, is also provided to execute the steps of a method of thepresent invention.

While the methods and apparatus have been described in connection witharrays of various moieties, such as polynucleotides or DNA, othermoieties can include any chemical moieties such as biopolymers.

The present invention can provide any one or more of the following orother benefits. For example, an array can be scanned such that loss ofsignal from saturation effects is kept low, or maintained at similarlevels during the array scanning process.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described with reference to thedrawings, in which:

FIG. 1 is a perspective view of a substrate carrying a typical array, asmay be used with, or part of, a package of the present invention;

FIG. 2 is an enlarged view of a portion of FIG. 1 showing some of theidentifiable individual regions of a single array of FIG. 1;

FIG. 3 is an enlarged cross-section of a portion of FIG. 2;

FIG. 4 is a front view of an array package in the form of a cartridge;

FIG. 5 illustrates a typical scanning beam spatial power distribution inrelation to array features;

FIGS. 6 and 7 illustrate conventional array scanning patterns;

FIG. 8–10 illustrate methods of the present invention; and

FIG. 11 illustrates an apparatus of the present invention.

To facilitate understanding, the same reference numerals have been used,where practical, to designate similar elements that are common to theFIGS.

DETAILED DESCRIPTION OF THE INVENTION

In the present application, unless a contrary intention appears, thefollowing terms refer to the indicated characteristics. A “biopolymer”is a polymer of one or more types of repeating units. Biopolymers aretypically found in biological systems and particularly includepolysaccharides (such as carbohydrates), and peptides (which term isused to include polypeptides and proteins) and polynucleotides as wellas their analogs such as those compounds composed of or containing aminoacid analogs or non-amino acid groups, or nucleotide analogs ornon-nucleotide groups. This includes polynucleotides in which theconventional backbone has been replaced with a non-naturally occurringor synthetic backbone, and nucleic acids (or synthetic or naturallyoccurring analogs) in which one or more of the conventional bases hasbeen replaced with a group (natural or synthetic) capable ofparticipating in Watson-Crick type hydrogen bonding interactions.Polynucleotides include single or multiple stranded configurations,where one or more of the strands may or may not be completely alignedwith another. A “nucleotide” refers to a sub-unit of a nucleic acid andhas a phosphate group, a 5 carbon sugar and a nitrogen containing base,as well as functional analogs (whether synthetic or naturally occurring)of such sub-units which in the polymer form (as a polynucleotide) canhybridize with naturally occurring polynucleotides in a sequencespecific manner analogous to that of two naturally occurringpolynucleotides. For example, a “biopolymer” includes DNA (includingcDNA), RNA, oligonucleotides, and PNA and other polynucleotides asdescribed in U.S. Pat. No. 5,948,902 and references cited therein (allof which are incorporated herein by reference), regardless of thesource. An “oligonucleotide” generally refers to a nucleotide multimerof about 10 to 100 nucleotides in length, while a “polynucleotide”includes a nucleotide multimer having any number of nucleotides. A“biomonomer” references a single unit, which can be linked with the sameor other biomonomers to form a biopolymer (for example, a single aminoacid or nucleotide with two linking groups one or both of which may haveremovable protecting groups). A biomonomer fluid or biopolymer fluidreference a liquid containing either a biomonomer or biopolymer,respectively (typically in solution).

An “array”, unless a contrary intention appears, includes any one-, two-or three-dimensional arrangement of addressable regions bearing aparticular chemical moiety or moieties (for example, biopolymers such aspolynucleotide sequences) associated with that region. An array is“addressable” in that it has multiple regions of different moieties (forexample, different polynucleotide sequences) such that a region (a“feature” or “spot” of the array) at a particular predetermined location(an “address”) on the array will detect a particular target or class oftargets (although a feature may incidentally detect non-targets of thatfeature). Array features are typically, but need not be, separated byintervening spaces. In the case of an array, the “target” will bereferenced as a moiety in a mobile phase (typically fluid), to bedetected by probes (“target probes”) which are bound to the substrate atthe various regions. However, either of the “target” or “target probes”may be the one which is to be evaluated by the other (thus, either onecould be an unknown mixture of polynucleotides to be evaluated bybinding with the other). An “array layout” refers collectively to one ormore characteristics of the features, such as feature positioning, oneor more feature dimensions, and some indication of a moiety at a givenlocation. “Hybridizing” and “binding”, with respect to polynucleotides,are used interchangeably.

When one item is indicated as being “remote” from another, this isreferenced that the two items are at least in different buildings, andmay be at least one mile, ten miles, or at least one hundred milesapart. “Communicating” information references transmitting the datarepresenting that information as electrical signals over a suitablecommunication channel (for example, a private or public network).“Forwarding” an item refers to any means of getting that item from onelocation to the next, whether by physically transporting that item orotherwise (where that is possible) and includes, at least in the case ofdata, physically transporting a medium carrying the data orcommunicating the data. An array “package” may be the array plus only asubstrate on which the array is deposited, although the package mayinclude other features (such as a housing with a chamber). A “chamber”references an enclosed volume (although a chamber may be accessiblethrough one or more ports). It will also be appreciated that throughoutthe present application, that words such as “top”, “upper”, and “lower”are used in a relative sense only. A “location” refers to any finitesmall area on the array that can be illuminated and any resultingfluorescence therefrom simultaneously (or shortly thereafter) detected,for example a pixel. “Timewise” and “temporally” are used synonymouslyto indicate relationship in time. Thus, events which are temporallysequential follow one after the other in time, whereas items which arespatially sequential follow one after the other in space. A “processor”references any combination of hardware or software which can controlcomponents as required to execute recited steps and includes, forexample, a general purpose digital microprocessor suitably programmed(for example, from a computer readable medium carrying necessary programcode or by communication from a remote location) to perform all of thesteps required of it, or any hardware or software combination which willperform those or equivalent steps. Reference to a singular item,includes the possibility that there are plural of the same itemspresent. All patents and other references cited in this application, areincorporated into this application by reference except insofar as whereany definitions in those references conflict with those of the presentapplication (in which case the definitions of the present applicationare to prevail).

Referring first to FIGS. 1–3, a contiguous planar transparent substrate10 carries multiple features 16 disposed across a first surface 11 a ofsubstrate 10 and separated by areas 13. Features 16 are disposed in apattern which defines the array. A second surface 11 b of substrate 10does not carry any features. Substrate 10 may be of any shape althoughthe remainder of the package of the present invention may need to beadapted accordingly. A typical array may contain at least ten features16, or at least 100 features, at least 1,000, at least 100,000 features,or more. All of the features 16 may be of different composition, or someor all could be the same. Each feature carries a predetermined moiety ormixture of moieties which in the case of FIGS. 1–3 is a polynucleotidehaving a particular sequence. This is illustrated schematically in FIG.3 where regions 16 are shown as carrying different polynucleotidesequences. Features 16 may have widths (that is, diameter, for a roundspot) in the range from a minimum of about 10 μm to a maximum of about1.0 cm. In embodiments where very small spot sizes or feature sizes aredesired, features 16 may have widths in the range of about 1.0 μm to 1.0mm, usually about 5.0 μm to 500 μm, and more usually about 10 μm to 200μm. Arrays of FIGS. 1–3 can be manufactured by in situ or depositionmethods as discussed above. In use, a feature can detect apolynucleotide of a complementary sequence by hybridizing to it, such aspolynucleotide 18 being detected by feature 16 a in FIG. 3 (the “*” onpolynucleotide 18 indicating a label such as a fluorescent label). Useof arrays to detect particular moieties in a sample (such as targetsequences) are well known.

Referring now to FIG. 4 an array package 30 may include a housing 34which has received substrate 10 adjacent an opening. Substrate 10 issealed (such as by the use of a suitable adhesive) to housing 34 arounda margin 38 with the second surface 11 b facing outward. Housing 34 isconfigured such that housing 34 and substrate 10, define a chamber intowhich features 16 of array 12 face. This chamber is accessible throughresilient septa 42, 50 which define normally closed ports of thechamber. Array package 30 preferably includes an identification (“ID”)54 of the array (sometimes referenced herein as an “identifier”). Theidentifier 54 may be in the form of a bar code or some other machinereadable code applied during the manufacture of array package 30.Identifier 54 may itself contain information on a saturationcharacteristic of the fluorescent label (a fluorophore) on targetpolynucleotide 18. This can be done, for example, either where it isknown that a target in a sample to which an array of the type of array12 is exposed, will likely contain a particular label, or where thearray 12 was associated with a known target label in the form of atarget labeling kit or associated with instructions to use a particularlabel. Throughout this application “association” of any these or otheritems with the array, can be accomplished, for example, by the itemsbeing present in the same package as the array when shipped to an enduser. In an alternative procedure, identifier 54 may be simply a uniqueseries of characters which is also stored in a local or remote databasein association with the foregoing label saturation characteristicinformation. Such a database may be established by an array manufacturerand communicated to the user (from a remote, or local source such as ona portable storage medium associated with the array) for retrieval inresponse to providing the identifier 54.

It will be appreciated though, that other array packages may be used.For example, the array package may consist only of the array of features16 on substrate 10 (in which case ID 54 can be applied directly tosubstrate 10). Thus, an array package need not include any housing orclosed chamber.

The components of the embodiments of the package 30 described above, maybe made of any suitable material. For example, housing 34 can be made ofmetal or plastic such as polypropylene, polyethylene oracrylonitrile-butadiene-styrene (“ABS”). Substrate 10 may be of anysuitable material, and is preferably sufficiently transparent to thewavelength of an interrogating and array emitted light, as to allowinterrogation without removal from housing 34. Such transparent andnon-transparent materials include, for flexible substrates: nylon, bothmodified and unmodified, nitrocellulose, polypropylene, and the like.For rigid substrates, specific materials of interest include: glass;fused silica, silicon, plastics (for example, polytetrafluoroethylene,polypropylene, polystyrene, polycarbonate, and blends thereof, and thelike); metals (for example, gold, platinum, and the like). The firstsurface 11 a of substrate 10 may be modified with one or more differentlayers of compounds that serve to modify the properties of the surfacein a desirable manner. Such modification layers, when present, willgenerally range in thickness from a monomolecular thickness to about 1mm, usually from a monomolecular thickness to about 0.1 mm and moreusually from a monomolecular thickness to about 0.001 mm. Modificationlayers of interest include: inorganic and organic layers such as metals,metal oxides, polymers, small organic molecules and the like. Polymericlayers of interest include layers of: peptides, proteins, polynucleicacids or mimetics thereof (for example, peptide nucleic acids and thelike); polysaccharides, phospholipids, polyurethanes, polyesters,polycarbonates, polyureas, polyamides, polyethyleneamines, polyarylenesulfides, polysiloxanes, polyimides, polyacetates, and the like, wherethe polymers may be hetero- or homopolymeric, and may or may not haveseparate functional moieties attached thereto (for example, conjugated),The materials from which substrate 10 and housing 34 (at least theportion facing toward the inside of chamber 36) may be fabricated shouldideally themselves exhibit a low level of binding during hybridizationor other events.

Referring to FIG. 5, a concept of the present invention is illustrated.In FIG. 5 power distribution 60 is a typical spatial power distributioncurve of an illumination source in the form of a laser beam of circularcross-section focused onto an array 12. Power distribution 60 has ahalf-height width 62 of typically about 1–10 μm with each square pixel70 detected by a detector having a width of from about one to ten timeshalf-height width 62. This also illustrates a difficulty withconventional beam scanning schemes. In particular, in a typical scanningconfiguration a laser beam of FIG. 5 may be used to scan a row of pixels70 a (extending in and out of the page) and any resulting fluorescencesignals from those pixels detected by a suitable detector.Conventionally the laser beam is then moved over a width of a pixel to anext row 70 b of pixels to be detected, and is then moved along that row(that is, in a direction in or out of the paper) and any resultingsignals detected. The pixel being detected by the detector is movedalong with the beam in a known manner or as described below. Thissimultaneous beam and detection movement (often referenced together as“scanning”, “scanned” and the like), is repeated moving from one row tothe next adjacent row until the entire array 12 has been covered.However, the present invention recognizes that when a given row ofpixels 70 is being scanned, other adjacent rows are being illuminated bythe laser beam (although perhaps with lower power than the row beingscanned and detected). For example, while row 70 a in FIG. 5 is beingscanned, 70 b, 70 e, 70 c, 70 d are also being simultaneously exposed tothe laser beam (although no resulting fluorescence signal is beingdetected from them). This means that some percentage of fluorescentmoieties in each of those adjacent rows will become lost to a tripletstate. When pixels of the next adjacent row, for example row 70 b, arescanned all of the fluorescent moieties therein may not have hadsufficient time to return from a triplet to ground state. Thus, whenthose adjacent rows are scanned in turn, some of the fluorescent signalhas already been lost to the triplet state (in addition to a portionwhich may be lost during the scanning of that adjacent row).

For ease of reference, as already pointed out “scanning” will often beused by itself in a manner which will be understood to include actuallymoving the illuminating source as needed and the detecting of theresulting fluorescence. Also, in FIGS. 6–10 the arrows indicate scanningdirections across rows of pixels 70 with suffixes “a”, “b”, “c” and thelike being used to indicate scanning order (the smaller case equivalentof “L” is not used as a suffix). Thus, in the conventional rectangularscanning pattern of FIG. 6 a row 70 a is first scanned from left toright as viewed in the FIG. 7, the laser beam is then re-positioned at anext adjacent row 70 b which is then scanned from right to left, then to70 c which is scanned from left to right, and so on.

Referring to FIGS. 6 and 7 conventional scanning patterns areillustrated. Note from FIG. 6 then, that as described in connection withFIG. 5 while one row 70 (such as 70 a) is being scanned, followed by anext adjacent row (such as 70 b), the next adjacent row (such as 70 b)is still being illuminated and some proportion of the species may belost to the triplet state and not have had time to recover (that is,return from the triplet to the ground state) by the time that adjacentrow (such as 70 b) is then scanned. Even worse, the proportion that isstill in the triplet state may vary along the length of a pixel rowleading to non-uniformity in signals from pixels which otherwise mightbe uniform. For example, when row 70 a is scanned from left to right,the rightmost pixel of row 70 b has less time to recover than theleftmost pixel of row 70 b, and thus the detected fluorescent signalfrom row 70 b may increase from right to left even if conditions alongrow 70 b are otherwise constant.

Referring to FIG. 7 a conventional zigzag scanning pattern isillustrate. In this pattern a row 70 a is scanned from left to right,the beam then re-positioned back to the left hand side of row 70 b toscan row 70 b also from left to right, and this process repeated for allrows of pixels 70 to be scanned on the array. The laser beam may beturned off while being re-positioned from one row to the next adjacentrow. Thus, in the zigzag scanning pattern all pixel rows are scanned ina same direction. While this pattern does not suffer from the sameuniformity concerns as the rectangular pattern, time is wasted movingthe laser from one end of one row back to the next end of an adjacentrow. For an array with many rows (for example, one hundred), this canamount to a large amount of wasted time. Furthermore, as in therectangular scanning pattern, some proportion of fluorophores in pixelsof the adjacent row are still lost to the triplet state since there maybe insufficient time for them to recover. In both FIGS. 6 and 7, thetimewise sequence of the linear scanned paths is the same as the spatialsequence. That is, if one moves across the lines 70 in the order inwhich they spatially occur (70 a, 70 b, 70 c, and so on), this is thesame as their timewise order.

Referring to FIG. 8, a method of the present invention is illustrated.In the scanning pattern of FIG. 8 multiple parallel lines of pixels 70on array 12 are illuminated by moving a focused laser beam across themand any resulting fluorescence detected (that is, the lines of pixels 70are scanned). However, unlike FIGS. 6 and 7 the timewise sequence of thelines 70 in FIG. 8 (or FIG. 9 or 10) is no longer the same as theirspatial sequence. In particular, in FIG. 8 it will be seen that multiplelater illuminated lines (such as lines 70 c, 70 e, 70 g, 70 i) are allcloser to an earlier illuminated line (such as line 70 a) than atemporally intervening illuminated line (such as line 70 b), and in factthose later illuminated lines (such as lines 70 c, 70 e, 70 g, 70 i) areinterleaved between the earlier (such as 70 a) and temporallyintervening (such as 70 b) illuminated lines. Note that in FIG. 8 afirst cycle of scanning can be considered lines 70 a through 70 j, witha next cycle started by lines 70 k, 70 m (lines subsequent to 70 m arerepeated in a same pattern as 70 c through 70 j). Note that in thisexample (and in FIGS. 9 and 10), the spacing between all scan lines(including the nearest lines of the earlier, temporally intervening andinterleaved lines) is equal. Also, it will be seen that in FIG. 8 (andin FIGS. 9 and 10) timewise successive scan lines of any cycle areilluminated scanned in opposite directions. However, while this oppositeconfiguration saves time, it is not essential and timewise successivescan lines could be scanned in the same direction. The laser beam can beturned off so as not to provide any illumination at the array while itis being re-positioned between lines.

FIG. 9 illustrates a further scanning method of the present invention,wherein lines 70 a through 70 k represent one scanning cycle which maythen be repeated starting in a new cycle with line 70 m (subsequentlines not being shown). Similarly, lines 70 a through 70 k in FIG. 10represent a cycle of a further scanning method of the present inventionwith lines 70 a through 70 j representing a first cycle and lines 70 k,70 m being the first two lines of a next cycle which repeats the samepattern of the first cycle. It should be appreciated that while scanningis performed along lines as shown in FIGS. 8–10, this is not necessary.For example, other patterns could be used the same as in FIGS. 8–10 butwherein instead of the lines 70 (each of which also has multiplelocations within it) could instead each be replaced with just a singlelocation (that is, just single pixels; in this situation those locationswould lie along a same line extending up and own as viewed in FIGS.8–10). Further, paths other than straight lines could be used (forexample, if array 12 had rows of semi-circular features, semi-circularscan paths could be used, although this would not be necessary).

Other scanning patterns within the present invention are possible. Forexample, assume spatially sequential rows on an array are numbered 1, 2,3, 4, 5, 6 and so on to n (where n is an integer), from a top of thearray down, and that L and R are used to indicate scanning from left toright, and right to left, respectively. One particular scanning patternwould include the following timewise sequence 1L, 6R, 3L, 8R, 5L, 10Rand so on (that is, row 1 is scanned from left to right, then row 6 fromright to left, then row three from left to right, then row 8 from rightto left and so on). The foregoing scanning sequence can be generallyrepresented as repeated cycles of scanning along row (2n−5)L followed byrow (2n)R (where n starts at an integer, in this case 3, such that eachscan line is a positive number), n increasing by 1 in each subsequentcycle. A variation of the foregoing then, would be (2n−9)L, (2n)R, ormore generally (2n−a)L, (2n)R where a is any positive odd integergreater than 3. Again, n begins with a positive integer such that (2n−a)is positive, and increments by 1 in each cycle.

It will be appreciated that many other scanning patterns are possiblewithin a method of the present invention. Which pattern may beconsidered best in a particular situation depends upon how much time isdesired to elapse before a line spatially adjacent a previously scannedline is to elapse. In particular, a desired time between illuminating aline and illuminating a spatially closest later illuminated line can beevaluated. The desired time will depend on factors such as scan speed(higher scan speeds suggest greater spatial distance between temporallyadjacent scan lines), laser beam power distribution at the array, pixelsize, and a saturation characteristic (that is, what proportion will beexcited to a triplet state and the rate of return therefrom to theground state) of a fluorophore producing the fluorescence. Suitable scanpatterns may then be calculated from known values, or can be readilydetermined experimentally. For example, using a test array which has auniform fluorophore distribution thereon, scan patterns can be testedwith increasing spatial distance between temporally adjacent scan linesuntil no further improvement in detected fluorescence signal isobserved.

Referring now to FIG. 11, an apparatus of the present invention (whichmay be generally referenced as an array “scanner”) is illustrated withan array package 30 mounted therein. A light system provides light froma laser 100 which passes through an electro-optic modulator (EOM) 110with attached polarizer 120. A control signal in the form of a variablevoltage applied to the EOM 110 by the controller (CU) 180 changes thepolarization of the exiting light which is thus more or less attenuatedby the polarizer 120. Controller 180 may be or include a suitablyprogrammed processor. Thus, EOM 110 and polarizer 120 together act as avariable optical attenuator which can alter the power of aninterrogating light spot exiting from the attenuator. The remainder ofthe light transmitted by beam splitter 140 is in this case reflected offa dichroic beam splitter 154 and focused onto the array 12 of arraypackage 30 using optical components in beam focuser 160. Light emittedfrom features 16 in response to the interrogating light, in particularfluorescence, is imaged, for example, using the same optics infocuser/scanner 160, and passes through the dichroic beamsplitter 154and onto a detector (PMT) 150. More optical components (not shown) maybe used between the dichroic and the PMT (lenses, pinholes, filters,fibers etc.) and the detector 150 may be of various different types(e.g. a photo-multiplier tube (PMT) or a CCD or an avalanche photodiode(APD)). A scanning system causes the interrogating light spot to bescanned across multiple sites on an array package 30 received in theapparatus, which sites include at least the multiple features 16 of thearray. In particular the scanning system is typically a line by linescanner, scanning the interrogating light in a line across the arraypackage 30, then moving (“transitioning”) the interrogating light tobegin scanning a timewise next row, scanning that timewise next row, andrepeating the foregoing procedure row after row in accordance with anyof the methods of the present invention (for example, any of thosepatterns in FIGS. 8–10). This can be accomplished by providing a housing164 containing mirror 154, focuser 160, and detector 150, which housing164 can be moved along a line of pixels (that is, from left to right orthe reverse as viewed in FIG. 11) by transporter 162. In practice,detector 150 may be stationary with further suitable optics (forexample, an additional mirror in housing 164) to allow this. A seconddirection of scanning can be provided by transporter 190 which movesarray package 30 one or more lines 70 in a direction in and out of paperas viewed in FIG. 11. Transporter 190 may use a same or differentactuator components to accomplish coarse (a larger number of lines)movement and finer movement (a smaller number of lines). For example, afirst actuator (such as a linear or stepper motor) could accomplishcoarse movement while a second actuator (such as a piezoelectric orelectromagnetic pusher) could be used to provide the finer movement (forexample, in a closed loop fashion or by driving between two mechanicalstops).

The apparatus of FIG. 11 may further include a reader 170 which reads anidentifier 54 from an array package 30. When identifier 54 is in theform of a bar code, reader 170 may be a suitable bar code reader.

Controller 180 of the apparatus is connected to receive fluorescentsignals emitted in response to the interrogating light from emittedsignal detector 130 and signals indicating a read identification fromreader 170, and to provide the control signal to EOM 110. Controller 180may also analyze, store, and/or output data relating to emitted signalsreceived from detector 130 in a known manner. Controller 180 may includea computer in the form of a programmable digital processor, and includea media reader 182 which can read a portable removable media (such as amagnetic or optical disk), and a communication module 184 which cancommunicate over a communication channel (such as a network, for examplethe internet or a telephone network) with a remote site (such as adatabase at which information relating to array package 30 may be storedin association with the identification 54). Controller 180 is suitablyprogrammed to execute all of the steps required by it during operationof the apparatus, as discussed further below. Alternatively, controller180 may be any hardware or hardware/software combination which canexecute those steps.

In one mode of operation, the array in package 30 is typically firstexposed to a liquid sample introduced into the chamber through one ofthe septa 42, 50. The array may then be washed and scanned with a liquid(such as a buffer solution) present in the chamber and in contact withthe array, or it may be dried following washing. Following a given arraypackage 30 being mounted in the apparatus, reader 170 automatically (orupon operator command) reads array ID 54. Controller 180 can then usethis ID 54 to retrieve information such as information which may aid inselecting a time between illuminating a line and illuminating aspatially closest later illuminated line based on an identifierassociated with the array. Such information may include saturationcharacteristics of a chromophore, and other information, as alreadymentioned. Such information may be retrieved directly from the contentsof ID 54 when ID 54 contains such information. Alternatively, ID 54 maybe used to retrieve such information from a database containing the IDin association with such information. Such a database may be a localdatabase accessible by controller 180 (such as may be contained in aportable storage medium in drive 182 which is associated with package30, such as by physical association with package 30 when received by theuser, or by a suitable identification), or may be a remote databaseaccessible by controller 180 through communication module 184 and asuitable communication channel (not shown).

Next, the scanning system scans timewise successive lines 70 of pixelsacross the array in any of the methods of the present invention. Duringsuch a row scan, the EOM 110 may be controlled by controller 180 to turnoff the illuminating light power at the array 12 during transitioningfrom line to line. This is repeated until the entire array 12 has beenscanned.

Results from a sample exposed array, read according to a method of thepresent invention, may be raw results (such as fluorescence intensityreadings for each feature in one or more color channels) or may beprocessed results such as obtained by rejecting a reading for a featurewhich is below a predetermined threshold and/or forming conclusionsbased on the pattern read from the array (such as whether or not aparticular target sequence may have been present in the sample). Theresults of the reading (processed or not) may be forwarded (such as bycommunication of data representing the results) to a remote location ifdesired, and received there for further use (such as furtherprocessing).

Note that a variety of geometries of the features 16 may be constructedother than the organized rows and columns of the array of FIGS. 1–3. Forexample, features 16 can be arranged in a series of curvilinear rowsacross the substrate surface (for example, a series of concentriccircles or semi-circles of spots), and the like. Even irregulararrangements of features 16 can be used, at least when some means isprovided such that during their use the locations of regions ofparticular characteristics can be determined (for example, a map of theregions is provided to the end user with the array). Furthermore,substrate 10 could carry more than one array 12, arranged in any desiredconfiguration on substrate 10. While substrate 10 is planar andrectangular in form, other shapes could be used with housing 34 beingadjusted accordingly. In many embodiments, substrate 10 will be shapedgenerally as a planar, rectangular solid, having a length in the rangeabout 4 mm to 200 mm, usually about 4 mm to 150 mm, more usually about 4mm to 125 mm; a width in the range about 4 mm to 200 mm, usually about 4mm to 120 mm and more usually about 4 mm to 80 mm; and a thickness inthe range about 0.01 mm to 5.0 mm, usually from about 0.1 mm to 2 mm andmore usually from about 0.2 to 1 mm. However, larger substrates can beused. Less preferably, substrate 10 could have three-dimensional shapewith irregularities in first surface 11 a. In any event, the dimensionsof housing 34 may be adjusted accordingly. Additionally, during scanningit is possible to illuminate all pixels of a line simultaneously (forexample, by using a line of light emitting diodes).

Various modifications to the particular embodiments described above are,of course, possible. Accordingly, the present invention is not limitedto the particular embodiments described in detail above.

1. An apparatus for reading fluorescence signals from an array ofchemical moieties, comprising: (a) an illumination source to causefluorescence of the chemical moieties; (b) a scan system to direct theillumination source to different locations on the array; and (c) adetector to detect any resulting fluorescence from the array; (c) aprocessor which controls the scan system such that multiple locations onthe array are illuminated and any resulting fluorescence detected,wherein a later illuminated location is spatially closer to an earlierilluminated location than is a temporally intervening illuminatedlocation lying on a same line as the later and earlier illuminatedlocations.
 2. An apparatus according to claim 1 wherein at least onelater illuminated location is interleaved between previously illuminatedlocations.
 3. An apparatus according to claim 1 wherein the processoradditionally selects a time between illuminating a line and illuminatinga spatially closest later illuminated line based on a saturationcharacteristic of a fluorophore producing the fluorescence.
 4. Anapparatus for reading fluorescence signals from an array of chemicalmoieties, comprising: (a) an illumination source to cause fluorescenceof the chemical moieties; (b) a scan system to direct the illuminationsource to different locations on the array; and (c) a detector to detectany resulting fluorescence; (c) a processor which controls the scansystem such that multiple paths across the array are illuminated and anyresulting fluorescence detected, wherein the paths extend in a samelengthwise direction and are spaced from one another in a crosswisedirection, and the spatial sequence of the paths does not correspond totheir temporal sequence.
 5. An apparatus according to claim 4 wherein atleast one later illuminated path is closer to a an earlier illuminatedpath than a temporally intervening illuminated path.
 6. An apparatusaccording to claim 5 wherein timewise successively illuminated paths areequally spaced crosswise from their respective closest later illuminatedpaths.
 7. An apparatus according to claim 5 wherein at least one laterilluminated path is interleaved between previously illuminated paths. 8.An apparatus according to claim 7 wherein multiple later illuminatedpaths are interleaved between previously illuminated paths.
 9. Anapparatus for reading fluorescence signals from an array of chemicalmoieties, comprising: (a) an illumination source to cause fluorescenceof the chemical moieties; (b) a scan system to direct the illuminationsource to different locations on the array; and (c) a detector to detectany resulting fluorescence from the array; (c) a processor whichcontrols the scan system such that multiple parallel lines across thearray are illuminated and any resulting fluorescence detected, wherein alater illuminated line is closer to an earlier illuminated line than atemporally intervening illuminated line.
 10. An apparatus according toclaim 9 wherein multiple later illuminated lines are interleaved betweenpreviously illuminated lines.
 11. An apparatus according to claim 10wherein the spacing between the interleaved and previously illuminatedlines is equal.
 12. An apparatus of claim 9 additionally comprisingrepeating the illuminating in one or more further cycles, and whereintimewise successively illuminated lines of a cycle are illuminated byscanning a light beam in opposite directions.
 13. An apparatus accordingto claim 9 wherein the processor additionally selects a time betweenilluminating a line and illuminating a spatially closest laterilluminated line based on a saturation characteristic of a fluorophoreproducing the fluorescence.
 14. An apparatus according to claim 9wherein the processor additionally selects a time between illuminating aline and illuminating a spatially closest later illuminated line basedon a spatial distribution of the illumination and a pixel size duringthe detecting.
 15. A computer program product, comprising: a computerreadable storage medium having a computer program stored thereon which,when loaded into a computer communicating with an apparatus for readingfluorescence signals from an array of chemical moieties, performs thesteps of: illuminating multiple locations on the array and detecting anyresulting fluorescence, wherein a later illuminated location isspatially closer to an earlier illuminated location than is a temporallyintervening illuminated location lying on a same line as the later andearlier illuminated locations.
 16. A computer program product accordingto claim 15 wherein at least one subsequently illuminated location isinterleaved between previously illuminated locations.
 17. A methodaccording to claim 16 additionally comprising selecting a time betweenilluminating a location and illuminating a spatially closest laterilluminated location based on a saturation characteristic of afluorophore producing the fluorescence.
 18. A computer program product,comprising: a computer readable storage medium having a computer programstored thereon which, when loaded into a computer communicating with anapparatus for reading fluorescence signals from an array of chemicalmoieties, performs the steps of: illuminating multiple parallel linesacross the array and detecting any resulting fluorescence from thearray, wherein a later illuminated line is closer to an earlierilluminated line than a temporally intervening illuminated line.
 19. Amethod according to claim 18 wherein each line comprises a series ofpoints illuminated sequentially by moving an illuminating beam along theline.